Respiratory syncytial virus infection during infancy and asthma during childhood in the USA (INSPIRE): a population-based, prospective birth cohort study.

BACKGROUND: Early-life severe respiratory syncytial virus (RSV) infection has been associated with the onset of childhood wheezing illnesses. However, the relationship between RSV infection during infancy and the development of childhood asthma is unclear. We aimed to assess the association between RSV infection during infancy and childhood asthma. METHODS: INSPIRE is a large, population-based, birth cohort of healthy infants with non-low birthweight born at term between June and December, 2012, or between June and December, 2013. Infants were recruited from 11 paediatric practices across middle Tennessee, USA. We ascertained RSV infection status (no infection vs infection) in the first year of life using a combination of passive and active surveillance with viral identification through molecular and serological techniques. Children were then followed up prospectively for the primary outcome of 5-year current asthma, which we analysed in all participants who completed 5-year follow-up. Statistical models, which were done for children with available data, were adjusted for child's sex, race and ethnicity, any breastfeeding, day-care attendance during infancy, exposure to second-hand smoke in utero or during early infancy, and maternal asthma. FINDINGS: Of 1946 eligible children who were enrolled in the study, 1741 (89%) had available data to assess RSV infection status in the first year of life. The proportion of children with RSV infection during infancy was 944 (54%; 95% CI 52-57) of 1741 children. The proportion of children with 5-year current asthma was lower among those without RSV infection during infancy (91 [16%] of 587) than those with RSV infection during infancy (139 [21%] of 670; p=0·016). Not being infected with RSV during infancy was associated with a 26% lower risk of 5-year current asthma than being infected with RSV during infancy (adjusted RR 0·74, 95% CI 0·58-0·94, p=0·014). The estimated proportion of 5-year current asthma cases that could be prevented by avoiding RSV infection during infancy was 15% (95% CI 2·2-26·8). INTERPRETATION: Among healthy children born at term, not being infected with RSV in the first year of life was associated with a substantially reduced risk of developing childhood asthma. Our findings show an age-dependent association between RSV infection during infancy and childhood asthma. However, to definitively establish causality, the effect of interventions that prevent, delay, or decrease the severity of the initial RSV infection on childhood asthma will need to be studied. FUNDING: US National Institutes of Health.

Effect of Infant RSV Infection on Memory T Cell Responses at Age 2-3 Years.

Background: It is unknown whether RSV infection in infancy alters subsequent RSV immune responses. Methods: In a nested cohort of healthy, term children, peripheral blood mononuclear cells (PBMCs) were collected at ages 2-3 years to examine RSV memory T cell responses among children previously RSV infected during infancy (first year of life) compared to those RSV-uninfected during infancy. The presence vs. absence of infant RSV infection was determined through a combination of RSV molecular and serologic testing. Memory responses were measured in RSV stimulated PBMCs. Results: Compared to children not infected with RSV during the first year of life, children infected with RSV during infancy had lower memory T cell responses at ages 2-3 years to in vitro stimulation with RSV for most tested type-1 and type-17 markers for a number of memory T cell subsets. Conclusions: RSV infection in infancy has long-term effects on memory T cell responses. This is the first study to show the potential for RSV infection in infancy to have long-term effects on the immune memory irrespective of the severity of the infection. Our results suggest a possible mechanism through which infant RSV infection may result in greater risk of subsequent childhood respiratory viral morbidity, findings also relevant to vaccine development.

Metabolic Reprogramming of Nasal Airway Epithelial Cells Following Infant Respiratory Syncytial Virus Infection.

Respiratory syncytial virus (RSV) is a seasonal mucosal pathogen that infects the ciliated respiratory epithelium and results in the most severe morbidity in the first six months of life. RSV is a common cause of acute respiratory infection during infancy and is an important early-life risk factor strongly associated with asthma development. While this association has been repeatedly demonstrated, limited progress has been made on the mechanistic understanding in humans of the contribution of infant RSV infection to airway epithelial dysfunction. An active infection of epithelial cells with RSV in vitro results in heightened central metabolism and overall hypermetabolic state; however, little is known about whether natural infection with RSV in vivo results in lasting metabolic reprogramming of the airway epithelium in infancy. To address this gap, we performed functional metabolomics, 13C glucose metabolic flux analysis, and RNA-seq gene expression analysis of nasal airway epithelial cells (NAECs) sampled from infants between 2-3 years of age, with RSV infection or not during the first year of life. We found that RSV infection in infancy was associated with lasting epithelial metabolic reprogramming, which was characterized by (1) significant increase in glucose uptake and differential utilization of glucose by epithelium; (2) altered preferences for metabolism of several carbon and energy sources; and (3) significant sexual dimorphism in metabolic parameters, with RSV-induced metabolic changes most pronounced in male epithelium. In summary, our study supports the proposed phenomenon of metabolic reprogramming of epithelial cells associated with RSV infection in infancy and opens exciting new venues for pursuing mechanisms of RSV-induced epithelial barrier dysfunction in early life.

Plasmacytoid dendritic cells produce type I interferon and reduce viral replication in airway epithelial cells after SARS-CoV-2 infection.

Infection with SARS-CoV-2 has caused a pandemic of unprecedented dimensions. SARS-CoV-2 infects airway and lung cells causing viral pneumonia. The importance of type I interferon (IFN) production for the control of SARS-CoV-2 infection is highlighted by the increased severity of COVID-19 in patients with inborn errors of type I IFN response or auto-antibodies against IFN-α. Plasmacytoid dendritic cells (pDCs) are a unique immune cell population specialized in recognizing and controlling viral infections through the production of high concentrations of type I IFN. In this study, we isolated pDCs from healthy donors and showed that pDCs are able to recognize SARS-CoV-2 and rapidly produce large amounts of type I IFN. Sensing of SARS-CoV-2 by pDCs was independent of viral replication since pDCs were also able to recognize UV-inactivated SARS-CoV-2 and produce type I IFN. Transcriptional profiling of SARS-CoV-2 and UV-SARS-CoV-2 stimulated pDCs also showed a rapid type I and III IFN response as well as induction of several chemokines, and the induction of apoptosis in pDCs. Moreover, we modeled SARS-CoV-2 infection in the lung using primary human airway epithelial cells (pHAEs) and showed that co-culture of pDCs with SARS-CoV-2 infected pHAEs induces an antiviral response and upregulation of antigen presentation in pHAE cells. Importantly, the presence of pDCs in the co-culture results in control of SARS-CoV-2 replication in pHAEs. Our study identifies pDCs as one of the key cells that can recognize SARS-CoV-2 infection, produce type I and III IFN and control viral replication in infected cells. IMPORTANCE: Type I interferons (IFNs) are a major part of the innate immune defense against viral infections. The importance of type I interferon (IFN) production for the control of SARS-CoV-2 infection is highlighted by the increased severity of COVID-19 in patients with defects in the type I IFN response. Interestingly, many cells are not able to produce type I IFN after being infected with SARS-CoV-2 and cannot control viral infection. In this study we show that plasmacytoid dendritic cells are able to recognize SARS-CoV-2 and produce type I IFN, and that pDCs are able to help control viral infection in SARS-CoV-2 infected airway epithelial cells.

CX3CR1 Engagement by Respiratory Syncytial Virus Leads to Induction of Nucleolin and Dysregulation of Cilia-related Genes.

Respiratory syncytial virus (RSV) contains a conserved CX3C motif on the ectodomain of the G-protein. The motif has been indicated as facilitating attachment of the virus to the host initiating infection via the human CX3CR1 receptor. The natural CX3CR1 ligand, CX3CL1, has been shown to induce signaling pathways resulting in transcriptional changes in the host cells. We hypothesize that binding of RSV to CX3CR1 via CX3C leads to transcriptional changes in host epithelial cells. Using transcriptomic analysis, the effect of CX3CR1 engagement by RSV was investigated. Normal human bronchial epithelial (NHBE) cells were infected with RSV virus containing either wildtype G-protein, or a mutant virus containing a CX4C mutation in the G-protein. RNA sequencing was performed on mock and 4-days-post-infected cultures. NHBE cultures were also treated with purified recombinant wild-type A2 G-protein. Here we report that RSV infection resulted in significant changes in the levels 766 transcripts. Many nuclear associated proteins were upregulated in the WT group, including nucleolin. Alternatively, cilia-associated genes, including CC2D2A and CFAP221 (PCDP1), were downregulated. The addition of recombinant G-protein to the culture lead to the suppression of cilia-related genes while also inducing nucleolin. Mutation of the CX3C motif (CX4C) reversed these effects on transcription decreasing nucleolin induction and lessening the suppression of cilia-related transcripts in culture. Furthermore, immunohistochemical staining demonstrated decreases in in ciliated cells and altered morphology. Therefore, it appears that engagement of CX3CR1 leads to induction of genes necessary for RSV entry as well as dysregulation of genes associated with cilia function.ImportanceRespiratory Syncytial Virus (RSV) has an enormous impact on infants and the elderly including increased fatality rates and potential for causing lifelong lung problems. Humans become infected with RSV through the inhalation of viral particles exhaled from an infected individual. These virus particles contain specific proteins that the virus uses to attach to human ciliated lung epithelial cells, initiating infection. Two viral proteins, G-protein and F-protein, have been shown to bind to human CX3CR1and nucleolin, respectively. Here we show that the G-protein induces nucleolin and suppresses gene transcripts specific to ciliated cells. Furthermore, we show that mutation of the CX3C-motif on the G-protein, CX4C, reverses these transcriptional changes.

Type I and Type III Interferons Restrict SARS-CoV-2 Infection of Human Airway Epithelial Cultures.

The newly emerged human coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has caused a pandemic of respiratory illness. Current evidence suggests that severe cases of SARS-CoV-2 are associated with a dysregulated immune response. However, little is known about how the innate immune system responds to SARS-CoV-2. In this study, we modeled SARS-CoV-2 infection using primary human airway epithelial (pHAE) cultures, which are maintained in an air-liquid interface. We found that SARS-CoV-2 infects and replicates in pHAE cultures and is directionally released on the apical, but not basolateral, surface. Transcriptional profiling studies found that infected pHAE cultures had a molecular signature dominated by proinflammatory cytokines and chemokine induction, including interleukin 6 (IL-6), tumor necrosis factor alpha (TNF-α), and CXCL8, and identified NF-κB and ATF-4 as key drivers of this proinflammatory cytokine response. Surprisingly, we observed a complete lack of a type I or III interferon (IFN) response to SARS-CoV-2 infection. However, pretreatment and posttreatment with type I and III IFNs significantly reduced virus replication in pHAE cultures that correlated with upregulation of antiviral effector genes. Combined, our findings demonstrate that SARS-CoV-2 does not trigger an IFN response but is sensitive to the effects of type I and III IFNs. Our studies demonstrate the utility of pHAE cultures to model SARS-CoV-2 infection and that both type I and III IFNs can serve as therapeutic options to treat COVID-19 patients.IMPORTANCE The current pandemic of respiratory illness, COVID-19, is caused by a recently emerged coronavirus named SARS-CoV-2. This virus infects airway and lung cells causing fever, dry cough, and shortness of breath. Severe cases of COVID-19 can result in lung damage, low blood oxygen levels, and even death. As there are currently no vaccines approved for use in humans, studies of the mechanisms of SARS-CoV-2 infection are urgently needed. Our research identifies an excellent system to model SARS-CoV-2 infection of the human airways that can be used to test various treatments. Analysis of infection in this model system found that human airway epithelial cell cultures induce a strong proinflammatory cytokine response yet block the production of type I and III IFNs to SARS-CoV-2. However, treatment of airway cultures with the immune molecules type I or type III interferon (IFN) was able to inhibit SARS-CoV-2 infection. Thus, our model system identified type I or type III IFN as potential antiviral treatments for COVID-19 patients.

In vitro model for the assessment of human immune responses to subunit RSV vaccines.

Respiratory syncytial virus (RSV) is the single most important cause of serious lower respiratory tract disease in infants and young children worldwide and a high priority for vaccine development. Despite over 50 years of research, however, no vaccine is yet available. One block to vaccine development is an incomplete understanding of the aberrant memory response to the formalin-inactivated RSV vaccine (FI-RSV) given to children in the 1960s. This vaccine caused enhanced respiratory disease (ERD) with later natural RSV infection. Concern that any non-live virus vaccine may also cause ERD has blocked development of subunit vaccines for young children. A number of animal FI-RSV studies suggest various immune mechanisms behind ERD. However, other than limited data from the original FI-RSV trial, there is no information on the human ERD-associated responses. An in vitro model with human blood specimens may shed light on the immune memory responses likely responsible for ERD. Memory T cell responses to an antigen are guided by the innate responses, particularly dendritic cells that present an antigen in conjunction with co-stimulatory molecules and cytokine signaling. Our in vitro model involves human monocyte derived dendritic cells (moDC) and allogenic T cell cultures to assess innate responses that direct T cell responses. Using this model, we evaluated human responses to live RSV, FI-RSV, and subunit RSV G vaccines (G-containing virus-like particles, G-VLP). Similar to findings in animal studies, FI-RSV induced prominent Th2/Th17-biased responses with deficient type-1 responses compared to live virus. Responses to G-VLPs were similar to live virus, i.e. biased towards a Th1 and not a Th2/Th17. Also mutating CX3C motif in G gave a more pronounced moDC responses associated with type-1 T cell responses. This in vitro model identifies human immune responses likely associated with ERD and provides another pre-clinical tool to assess the safety of RSV vaccines.

Biology of Infection and Disease Pathogenesis to Guide RSV Vaccine Development.

Respiratory syncytial virus (RSV) is a leading cause of severe lower respiratory tract disease in young children and a substantial contributor to respiratory tract disease throughout life and as such a high priority for vaccine development. However, after nearly 60 years of research no vaccine is yet available. The challenges to developing an RSV vaccine include the young age, 2-4 months of age, for the peak of disease, the enhanced RSV disease associated with the first RSV vaccine, formalin-inactivated RSV with an alum adjuvant (FI-RSV), and difficulty achieving protection as illustrated by repeat infections with disease that occur throughout life. Understanding the biology of infection and disease pathogenesis has and will continue to guide vaccine development. In this paper, we review the roles that RSV proteins play in the biology of infection and disease pathogenesis and the corresponding contribution to live attenuated and subunit RSV vaccines. Each of RSV's 11 proteins are in the design of one or more vaccines. The G protein's contribution to disease pathogenesis through altering host immune responses as well as its role in the biology of infection suggest it can make a unique contribution to an RSV vaccine, both live attenuated and subunit vaccines. One of G's potential unique contributions to a vaccine is the potential for anti-G immunity to have an anti-inflammatory effect independent of virus replication. Though an anti-viral effect is essential to an effective RSV vaccine, it is important to remember that the goal of a vaccine is to prevent disease. Thus, other effects of the infection, such as G's alteration of the host immune response may provide opportunities to induce responses that block this effect and improve an RSV vaccine. Keeping in mind the goal of a vaccine is to prevent disease and not virus replication may help identify new strategies for other vaccine challenges, such as improving influenza vaccines and developing HIV vaccines.

Mutation of Respiratory Syncytial Virus G Protein's CX3C Motif Attenuates Infection in Cotton Rats and Primary Human Airway Epithelial Cells.

Despite being a high priority for vaccine development, no vaccine is yet available for respiratory syncytial virus (RSV). A live virus vaccine is the primary type of vaccine being developed for young children. In this report, we describe our studies of infected cotton rats and primary human airway epithelial cells (pHAECs) using an RSV r19F with a mutation in the CX3C chemokine motif in the RSV G protein (CX4C). Through this CX3C motif, RSV binds to the corresponding chemokine receptor, CX3CR1, and this binding contributes to RSV infection of pHAECs and virus induced host responses that contribute to disease. In both the cotton rat and pHAECs, the CX4C mutation decreased virus replication and disease and/or host responses to infection. Thus, this mutation, or other mutations that block binding to CX3CR1, has the potential to improve a live attenuated RSV vaccine by attenuating both infection and disease pathogenesis.

The Morphology and Assembly of Respiratory Syncytial Virus Revealed by Cryo-Electron Tomography.

Human respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract disease in young children. With repeat infections throughout life, it can also cause substantial disease in the elderly and in adults with compromised cardiac, pulmonary and immune systems. RSV is a pleomorphic enveloped RNA virus in the Pneumoviridae family. Recently, the three-dimensional (3D) structure of purified RSV particles has been elucidated, revealing three distinct morphological categories: spherical, asymmetric, and filamentous. However, the native 3D structure of RSV particles associated with or released from infected cells has yet to be investigated. In this study, we have established an optimized system for studying RSV structure by imaging RSV-infected cells on transmission electron microscopy (TEM) grids by cryo-electron tomography (cryo-ET). Our results demonstrate that RSV is filamentous across several virus strains and cell lines by cryo-ET, cryo-immuno EM, and thin section TEM techniques. The viral filament length varies from 0.5 to 12 µm and the average filament diameter is approximately 130 nm. Taking advantage of the whole cell tomography technique, we have resolved various stages of RSV assembly. Collectively, our results can facilitate the understanding of viral morphogenesis in RSV and other pleomorphic enveloped viruses.

Preexisting Immunity, More Than Aging, Influences Influenza Vaccine Responses.

Background. Influenza disproportionately impacts older adults while current vaccines have reduced effectiveness in the older population. Methods. We conducted a comprehensive evaluation of cellular and humoral immune responses of adults aged 50 years and older to the 2008-2009 seasonal trivalent inactivated influenza vaccine and assessed factors influencing vaccine response. Results. Vaccination increased hemagglutination inhibition and neutralizing antibody; however, 66.3% of subjects did not reach hemagglutination inhibition titers ≥ 40 for H1N1, compared with 22.5% for H3N2. Increasing age had a minor negative impact on antibody responses, whereas prevaccination titers were the best predictors of postvaccination antibody levels. Preexisting memory B cells declined with age, especially for H3N2. However, older adults still demonstrated a significant increase in antigen-specific IgG(+) and IgA(+) memory B cells postvaccination. Despite reduced frequency of preexisting memory B cells associated with advanced age, fold-rise in memory B cell frequency in subjects 60+ was comparable to subjects age 50-59. Conclusions. Older adults mounted statistically significant humoral and cell-mediated immune responses, but many failed to reach hemagglutination inhibition titers ≥40, especially for H1N1. Although age had a modest negative effect on vaccine responses, prevaccination titers were the best predictor of postvaccination antibody levels, irrespective of age.

CX3CR1 is an important surface molecule for respiratory syncytial virus infection in human airway epithelial cells.

Respiratory syncytial virus (RSV) is a major cause of severe pneumonia and bronchiolitis in infants and young children, and causes disease throughout life. Understanding the biology of infection, including virus binding to the cell surface, should help develop antiviral drugs or vaccines. The RSV F and G glycoproteins bind cell surface heparin sulfate proteoglycans (HSPGs) through heparin-binding domains. The G protein also has a CX3C chemokine motif which binds to the fractalkine receptor CX3CR1. G protein binding to CX3CR1 is not important for infection of immortalized cell lines, but reportedly is so for primary human airway epithelial cells (HAECs), the primary site for human infection. We studied the role of CX3CR1 in RSV infection with CX3CR1-transfected cell lines and HAECs with variable percentages of CX3CR1-expressing cells, and the effect of anti-CX3CR1 antibodies or a mutation in the RSV CX3C motif. Immortalized cells lacking HSPGs had low RSV binding and infection, which was increased markedly by CX3CR1 transfection. CX3CR1 was expressed primarily on ciliated cells, and ∼50 % of RSV-infected cells in HAECs were CX3CR1+. HAECs with more CX3CR1-expressing cells had a proportional increase in RSV infection. Blocking G binding to CX3CR1 with anti-CX3CR1 antibody or a mutation in the CX3C motif significantly decreased RSV infection in HAECs. The kinetics of cytokine production suggested that the RSV/CX3CR1 interaction induced RANTES (regulated on activation normal T-cell expressed and secreted protein), IL-8 and fractalkine production, whilst it downregulated IL-15, IL1-RA and monocyte chemotactic protein-1. Thus, the RSV G protein/CX3CR1 interaction is likely important in infection and infection-induced responses of the airway epithelium, the primary site of human infection.

An anti-G protein monoclonal antibody treats RSV disease more effectively than an anti-F monoclonal antibody in BALB/c mice.

Respiratory syncytial virus (RSV) belongs to the family Paramyxoviridae and is the single most important cause of serious lower respiratory tract infections in young children, yet no highly effective treatment or vaccine is available. To clarify the potential for an anti-G mAb, 131-2G which has both anti-viral and anti-inflammatory effects, to effectively treat RSV disease, we determined the kinetics of its effect compared to the effect of the anti-F mAb, 143-6C on disease in mice. Treatment administered three days after RSV rA2-line19F (r19F) infection showed 131-2G decreased breathing effort, pulmonary mucin levels, weight loss, and pulmonary inflammation earlier and more effectively than treatment with mAb 143-6C. Both mAbs stopped lung virus replication at day 5 post-infection. These data show that, in mice, anti-G protein mAb is superior to treating disease during RSV infection than an anti-F protein mAb similar to Palivizumab. This combination of anti-viral and anti-inflammatory activity makes 131-2G a promising candidate for treating for active human RSV infection.

Prophylaxis with a respiratory syncytial virus (RSV) anti-G protein monoclonal antibody shifts the adaptive immune response to RSV rA2-line19F infection from Th2 to Th1 in BALB/c mice.

UNLABELLED: Respiratory syncytial virus (RSV) is the single most important cause of serious lower respiratory tract infections in young children, yet no highly effective treatment or vaccine is available. In the present study, we investigated the effect of prophylactic treatment with the intact and F(ab')2 forms of an anti-G protein monoclonal antibody (MAb), 131-2G, on the humoral and cellular adaptive immune responses to RSV rA2-line19F (r19F) challenge in BALB/c mice. The F(ab')2 form of 131-2G does not decrease virus replication, but intact 131-2G does. The serum specimens for antibodies and spleen cells for memory T cell responses to RSV antigens were analyzed at 30, 45, 75, and 95 days postinfection (p.i.) with or without prior treatment with 131-2G. The ratios of Th2 to Th1 antibody isotypes at each time p.i indicated that both forms of MAb 131-2G shifted the subclass response from a Th2 (IgG1 and IgG2b) to a Th1 (IgG2A) bias. The ratio of IgG1 to IgG2A antibody titer was 3-fold to 10-fold higher for untreated than MAb-treated mice. There was also some increase in IgG (22% ± 13% increase) and neutralization (32% increase) in antibodies with MAb 131-2G prophylaxis at 75 days p.i. Treatment with 131-2G significantly (P ≤ 0.001) decreased the percentage of interleukin-4 (IL-4)-positive CD4 and CD8 cells in RSV-stimulated spleen cells at all times p.i., while the percentage of interferon gamma (IFN-γ) T cells significantly (P ≤ 0.001) increased ≥ 75 days p.i. The shift from a Th2- to a Th1-biased T cell response in treated compared to untreated mice likely was directed by the much higher levels of T-box transcription factor (T-bet) (≥ 45% versus <10%) in CD4 and CD8 T cells and lower levels of Gata-3 (≤ 2% versus ≥ 6%) in CD4 T cells in peptide-stimulated, day 75 p.i. spleen cells. These data show that the RSV G protein affects both humoral and cellular adaptive immune responses, and induction of 131-2G-like antibodies might improve the safety and long-term efficacy of an RSV vaccine. IMPORTANCE: The data in this report suggest that the RSV G protein not only contributes to disease but also dampens the host immune response to infection. Both effects of G likely contribute to difficulties in achieving an effective vaccine. The ability of MAb 131-2G to block these effects of G suggests that inducing antibodies similar to 131-2G should prevent disease and enhance the adaptive immune response with later RSV infection. The fact that 131-2G binds to the 13-amino-acid region conserved among all strains and that flanking sequences are conserved within group A or group B strains simplifies the task of developing a vaccine to induce 131-2G-like antibodies. If our findings in mice apply to humans, then including the 131-2G binding region of G in a vaccine should improve its safety and efficacy.

Respiratory syncytial virus G protein CX3C motif impairs human airway epithelial and immune cell responses.

Respiratory syncytial virus (RSV) is a major cause of severe lower respiratory infection in infants and young children and causes disease in the elderly and persons with compromised cardiac, pulmonary, or immune systems. Despite the high morbidity rates of RSV infection, no highly effective treatment or vaccine is yet available. The RSV G protein is an important contributor to the disease process. A conserved CX3C chemokine-like motif in G likely contributes to the pathogenesis of disease. Through this motif, G protein binds to CX3CR1 present on various immune cells and affects immune responses to RSV, as has been shown in the mouse model of RSV infection. However, very little is known of the role of RSV CX3C-CX3CR1 interactions in human disease. In this study, we use an in vitro model of human RSV infection comprised of human peripheral blood mononuclear cells (PBMCs) separated by a permeable membrane from human airway epithelial cells (A549) infected with RSV with either an intact CX3C motif (CX3C) or a mutated motif (CX4C). We show that the CX4C virus induces higher levels of type I/III interferon (IFN) in A549 cells, increased IFN-α and tumor necrosis factor alpha (TNF-α) production by human plasmacytoid dendritic cells (pDCs) and monocytes, and increased IFN-γ production in effector/memory T cell subpopulations. Treatment of CX3C virus-infected cells with the F(ab')2 form of an anti-G monoclonal antibody (MAb) that blocks binding to CX3CR1 gave results similar to those with the CX4C virus. Our data suggest that the RSV G protein CX3C motif impairs innate and adaptive human immune responses and may be important to vaccine and antiviral drug development.

A respiratory syncytial virus (RSV) anti-G protein F(ab')2 monoclonal antibody suppresses mucous production and breathing effort in RSV rA2-line19F-infected BALB/c mice.

Respiratory syncytial virus (RSV) belongs to the family Paramyxoviridae and is the single most important cause of serious lower respiratory tract infections in young children, yet no highly effective treatment or vaccine is available. Increased airway resistance and increased airway mucin production are two manifestations of RSV infection in children. RSV rA2-line19F infection induces pulmonary mucous production and increased breathing effort in BALB/c mice and provides a way to assess these manifestations of RSV disease in an animal model. In the present study, we investigated the effect of prophylactic treatment with the F(ab')2 form of the anti-G protein monoclonal antibody (MAb) 131-2G on disease in RSV rA2-line19F-challenged mice. F(ab')2 131-2G does not affect virus replication. It and the intact form that does decrease virus replication prevented increased breathing effort and airway mucin production, as well as weight loss, pulmonary inflammatory-cell infiltration, and the pulmonary substance P and pulmonary Th2 cytokine levels that occur in mice challenged with this virus. These data suggest that the RSV G protein contributes to prominent manifestations of RSV disease and that MAb 131-2G can prevent these manifestations of RSV disease without inhibiting virus infection.

Rapid differentiation of monocytes into type I IFN-producing myeloid dendritic cells as an antiviral strategy against influenza virus infection.

Myeloid dendritic cells (mDCs) have long been thought to function as classical APCs for T cell responses. However, we demonstrate that influenza viruses induce rapid differentiation of human monocytes into mDCs. Unlike the classic mDCs, the virus-induced mDCs failed to upregulate DC maturation markers and were unable to induce allogeneic lymphoproliferation. Virus-induced mDCs secreted little, if any, proinflammatory cytokines; however, they secreted a substantial amount of chemoattractants for monocytes (MCP-1 and IP-10). Interestingly, the differentiated mDCs secreted type I IFN and upregulated the expression of IFN-stimulated genes (tetherin, IFITM3, and viperin), as well as cytosolic viral RNA sensors (RIG-I and MDA5). Additionally, culture supernatants from virus-induced mDCs suppressed the replication of virus in vitro. Furthermore, depletion of monocytes in a mouse model of influenza infection caused significant reduction of lung mDC numbers, as well as type I IFN production in the lung. Consequently, increased lung virus titer and higher mortality were observed. Taken together, our results demonstrate that the host responds to influenza virus infection by initiating rapid differentiation of circulating monocytes into IFN-producing mDCs, which contribute to innate antiviral immune responses.

Immunosenescence and Challenges of Vaccination against Influenza in the Aging Population.

Influenza is an important contributor to morbidity and mortality worldwide. Accumulation of genetic mutations termed antigenic drift, allows influenza viruses to inflict yearly epidemics that may result in 250,000 to 500,000 deaths annually. Over 90% of influenza-related deaths occur in the older adult population. This is at least in part a result of increasing dysregulation of the immune system with age, termed immunosenescence. This dysregulation results in reduced capacity to cope with infections and decreased responsiveness to vaccination. The older adult population is in dire need of improved vaccines capable of eliciting protective responses in the face of a waning immune system. This review focuses on the status of immunity, responses to influenza vaccination, and strategies that are currently being explored to elicit enhanced immune responses in this high risk population.

B- and T-cell memory elicited by a seasonal live attenuated reassortant influenza vaccine: assessment of local antibody avidity and virus-specific memory T-cells using trogocytosis-based method.

PURPOSE: The main purpose of vaccination is to generate immunological memory providing enhanced immune responses against infectious pathogens. The standard and most commonly used assay for influenza vaccine immunogenicity evaluation is a hemagglutination inhibition assay (HAI). It is clear now that HAI assay is unable to properly assess the proven protective immunity elicited by live attenuated influenza vaccines (LAIV). New methods need to be developed for more accurate LAIV immunogenicity assessment and prediction of vaccine efficacy among target populations. OBJECTIVE: Randomized placebo-controlled study of memory B- and T-cell responses to intranasal LAIV in young adults. METHODS: A total of 56 healthy young adults 18-20 years old received seasonal monovalent LAIV. Mucosal memory B-cell responses were measured by IgA avidity assessment in nasal swabs. CD4 memory T cells in peripheral blood were examined by the expression of CD45RO marker and in functional test by the ability of virus-specific T cells to maintain the trogocytosis with antigen-loaded target cells. RESULTS: Intranasal LAIV immunization enhances mucosal IgA avidity even without reliable increases in antibody titers. At the day 21 after vaccination, up to 40% of subjects demonstrated significant increases in both total and virus-specific CD4 memory T cells that were observed regardless of seroconversion rate measured by HAI assay. CONCLUSION: The data suggest that immunogenicity of LAIV vaccines should be evaluated on the mucosal and cellular immunity basis. The assays applied could be used to support influenza clinical trials through preliminary screening of volunteers and subsequent measurement of anti-influenza in immunity.

Improving immunogenicity and effectiveness of influenza vaccine in older adults.

Aging is associated with a decline in immune function (immunosenescence) that leads to progressive deterioration in both innate and adaptive immune functions. These changes contribute to the subsequent increased risk for infectious diseases and their sequelae. Vaccination is the most effective and inexpensive public health strategy for prevention of infection, despite the decreased efficacy of vaccines in older adults due to immunosenescence. The rapid rise in the older adult population globally represents a great challenge for vaccination programs. This article first addresses the status of innate and adaptive immune functions in aging and then focuses on influenza vaccine. The development history of influenza vaccines, current status, and potential strategies to improve the immunogenicity and vaccine effectiveness in older adults are discussed.